George A. Wilkinson

Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis

The formation and guidance of specialized endothelial tip cells is essential for both developmental and pathological angiogenesis. Notch-1 signalling regulates the generation of tip cells, which respond to gradients of vascular endothelial growth factor (VEGF-A). The molecular cues and signalling pathways that control the guidance of tip cells are poorly understood. Bidirectional signalling by Eph receptors and ephrin ligands represents one of the most important guidance cues involved in axon path finding. Here we show that ephrin-B2 reverse signalling involving PDZ interactions regulates endothelial tip cell guidance to control angiogenic sprouting and branching in physiological and pathological angiogenesis. In vivo, ephrin-B2 PDZ-signalling-deficient mice (ephrin-B2ΔV) exhibit a reduced number of tip cells with fewer filopodial extensions at the vascular front in the mouse retina. In pathological settings, impaired PDZ signalling decreases tumour vascularization and growth. Mechanistically, we show that ephrin-B2 controls VEGF receptor (VEGFR)-2 internalization and signalling. Importantly, internalization of VEGFR2 is necessary for activation and downstream signalling of the receptor and is required for VEGF-induced tip cell filopodial extension. Together, our results suggest that ephrin-B2 at the tip cell filopodia regulates the proper spatial activation of VEGFR2 endocytosis and signalling to direct filopodial extension. Blocking ephrin-B2 reverse signalling may be an attractive alternative or combinatorial anti-angiogenic therapy strategy to disrupt VEGFR2 function in tumour angiogenesis.

Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity.

During development, Eph receptors mediate the repulsive axon guidance function of ephrins, a family of membrane attached ligands with their own receptor-like signaling potential. In cultured glutamatergic neurons, EphB2 receptors were recently shown to associate with NMDA receptors at synaptic sites and were suggested to play a role in synaptogenesis. Here we show that Eph receptor stimulation in cultured neurons modulates signaling pathways implicated in synaptic plasticity, suggesting cross-talk with NMDA receptor-activated pathways. Mice lacking EphB2 have normal hippocampal synapse morphology, but display defects in synaptic plasticity. In EphB2(-/-) hippocampal slices, protein synthesis-dependent long-term potentiation (LTP) was impaired, and two forms of synaptic depression were completely extinguished. Interestingly, targeted expression of a carboxy-terminally truncated form of EphB2 rescued the EphB2 null phenotype, indicating that EphB2 kinase signaling is not required for these EphB2-mediated functions.

A noncoding antisense RNA in tie-1 locus regulates tie-1 function in vivo

Recently, messenger RNAs in eukaryotes have shown to associate with antisense (AS) transcript partners that are often referred to as long noncoding RNAs (lncRNAs) whose function is largely unknown. Here, we have identified a natural AS transcript for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1), tie-1AS lncRNA in zebrafish, mouse, and humans. In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript and in additional locations (ear and brain). The tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1 transcript levels, resulting in specific defects in endothelial cell contact junctions in vivo and in vitro. The ratio of tie-1 versus tie-1AS lncRNA is altered in human vascular anomaly samples. These results directly implicate noncoding RNA-mediated transcriptional regulation of gene expression as a fundamental control mechanism for physiologic processes, such as vascular development.

Tyrosine phosphorylation sites in ephrinB2 are required for hippocampal long-term potentiation but not long-term depression.

Long-lasting changes in synaptic function are thought to be the cellular basis for learning and memory and for activity-dependent plasticity during development. Long-term potentiation (LTP) and long-term depression (LTD) are two opposing forms of synaptic plasticity that help fine tune neural connections and possibly serve to store information in the brain. Eph receptor tyrosine kinases and their transmembrane ligands, the ephrinBs, have essential roles in certain forms of synaptic plasticity. At the CA3–CA1 hippocampal synapse, EphB2 and EphA4 receptors are critically involved in long-term plasticity independent of their cytoplasmic domains, suggesting that ephrinBs are the active signaling partners. In cell-based assays, ephrinB reverse signaling was previously shown to involve phosphotyrosine-dependent and postsynaptic density-95/Discs large/zona occludens-1 (PDZ) domain interaction-dependent pathways. Which reverse signaling mode is required at hippocampal synapses is unknown. To address this question, we used knock-in mice expressing mutant isoforms of ephrinB2 that are deficient in specific aspects of reverse signaling. Our analysis revealed that tyrosine phosphorylation sites in ephrinB2 are required to mediate normal hippocampal LTP, but not for LTD. Conversely, ephrinB2 lacking the C-terminal PDZ interaction site, but competent to undergo tyrosine phosphorylation, cannot mediate either form of long-term plasticity. Our results provide the first evidence for phosphotyrosine-dependent ephrinB reverse signaling in a neuronal network and for differential ephrinB2 reverse signaling in two forms of synaptic plasticity.

Nogo-B receptor is essential for angiogenesis in zebrafish via Akt pathway.

Our previous work has shown that axon guidance gene family Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro. To investigate NogoB-NgBR function in vivo, we cloned the zebrafish ortholog of both genes and studied loss of function in vivo using morpholino antisense technology. Zebrafish ortholog of Nogo-B is expressed in somite while expression of zebrafish NgBR is localized in intersomitic vessel (ISV) and axial dorsal aorta during embryonic development. NgBR or Nogo-B knockdown embryos show defects in ISV sprouting in the zebrafish trunk. Mechanistically, we found that NgBR knockdown not only abolished its ligand Nogo-B-stimulated endothelial cell migration but also reduced the vascular endothelial growth factor (VEGF)-stimulated phosphorylation of Akt and vascular endothelial growth factor-induced chemotaxis and morphogenesis of human umbilical vein endothelial cells. Further, constitutively activated Akt (myristoylated [myr]Akt) or human NgBR can rescue the NgBR knockdown umbilical vein endothelial cell migration defects in vitro or NgBR morpholino-caused ISV defects in vivo. These data place Akt at the downstream of NgBR in both Nogo-B- and VEGF-coordinated sprouting of ISVs. In summary, this study identifies the in vivo functional role for Nogo-B and its receptor (NgBR) in angiogenesis in zebrafish.

Endothelial cell–specific chemotaxis receptor (ecscr) promotes angioblast migration during vasculogenesis and enhances VEGF receptor sensitivity

Endothelial cell-specific chemotaxis receptor (ECSCR) is a cell surface protein expressed by blood endothelial cells with roles in endothelial cell migration and signal transduction. We investigated the function of ecscr in the development of the zebrafish vasculature. Zebrafish ecscr is expressed in angioblasts and in axial vessels during angioblast migration and vasculogenesis. Morpholino-directed ecscr knockdown resulted in defective angioblast migration in the posterior lateral plate mesoderm, a process known to depend on vascular endothelial-derived growth factor (VEGF). In cultured cells, transfected ECSCR localized to actin-rich membrane protrusions, colocalizing with kinase insert domain protein receptor (KDR)/VEGF receptor 2 in these regions. ECSCR-silenced cells show reduced VEGF-induced phosphorylation of KDR but not of FMS-like tyrosine kinase 1 (FLT1)/VEGF receptor 1. Finally, chemical inhibition of VEGF receptor activity in zebrafish resulted in angioblast deficiencies that partially overlap with those seen in ecscr morphants. We propose that ecscr promotes migration of zebrafish angioblasts by enhancing endothelial kdr sensitivity to VEGF.

Role for EphrinB2 in postnatal lung alveolar development and elastic matrix integrity

Alveoli are formed in the lung by the insertion of secondary tissue folds, termed septa, which are subsequently remodeled to form the mature alveolar wall. Secondary septation requires interplay between three cell types: endothelial cells forming capillaries, contractile interstitial myofibroblasts, and epithelial cells. Here, we report that postnatal lung alveolization critically requires ephrinB2, a ligand for Eph receptor tyrosine kinases expressed by the microvasculature. Mice homozygous for the hypomorphic knockin allele ephrinB2ΔV/ΔV, encoding mutant ephrinB2 with a disrupted C‐terminal PDZ interaction motif, show severe postnatal lung defects including an almost complete absence of lung alveoli and abnormal and disorganized elastic matrix. Lung alveolar formation is not sensitive to loss of ephrinB2 cytoplasmic tyrosine phosphorylation sites. Postnatal day 1 mutant lungs show extracellular matrix alterations without differences in proportions of major distal cell populations. We conclude that lung alveolar formation relies on endothelial ephrinB2 function. Developmental Dynamics 237:2220–2234, 2008.

Altered Expression Patterns of EphrinB2 and EphB2 in Human Umbilical Vessels and Congenital Venous Malformations

Vascular malformations cause discomfort and pain in children and are often associated with skeletal hypertrophy. Their molecular basis is poorly understood. Ephrin ligands and Eph receptor tyrosine kinases are involved in embryonic vascular development. In mice, some ephrin/Eph family members show a complementary expression pattern in blood vessels, with ephrinB2 being expressed on arterial and EphB4 on venous endothelium. Targeted deletions of the genes reveal their essential roles for conduit vessel development in mice, suggesting similar functions during human vascular development and deregulation in vascular malformations. Here, we have defined the expression patterns of human ephrinB2, EphB4, and EphB2 in normal vessels of neonates (i.e. umbilici) and adults and compared them with those in congenital venous malformations. In adults, normal vessels of the skin, muscle, and legs express ephrinB2 and EphB2 on arterial endothelial cells (ECs), whereas EphB4 is found in arteries and veins. In the umbilicus, EphB2 is a specific marker of arterial ECs, whereas ephrinB2 is additionally expressed in venous ECs, suggesting an arterial function of the veins. In venous malformations, the expression of EphB4 is not altered, but both ephrinB2 and EphB2 are ectopically expressed in venous ECs. This may reflect a nonphysiologic arterialization of malformed veins. Our study shows that the arterial markers ephrin B2 and EphB2 are expressed in a subset of veins, and it remains to be studied whether this is cause or consequence of an altered vascular identity.

Mmp17b Is Essential for Proper Neural Crest Cell Migration In Vivo

The extracellular matrix plays a critical role in neural crest (NC) cell migration. In this study, we characterize the contribution of the novel GPI-linked matrix metalloproteinase (MMP) zebrafish mmp17b. Mmp17b is expressed post-gastrulation in the developing NC. Morpholino inactivation of mmp17b function, or chemical inhibition of MMP activity results in aberrant NC cell migration with minimal change in NC proliferation or apoptosis. Intriguingly, a GPI anchored protein with metalloproteinase inhibitor properties, Reversion-inducing-Cysteine-rich protein with Kazal motifs (RECK), which has previously been implicated in NC development, is expressed in close apposition to NC cells expressing mmp17b, raising the possibility that these two gene products interact. Consistent with this possibility, embryos silenced for mmp17b show defective development of the dorsal root ganglia (DRG), a crest-derived structure affected in RECK mutant fish sensory deprived (sdp). Taken together, this study has identified the first pair of MMP, and their putative MMP inhibitor RECK that functions together in NC cell migration.

Fli+ etsrp+ Hemato-Vascular Progenitor Cells Proliferate at the Lateral Plate Mesoderm during Vasculogenesis in Zebrafish

Background Vasculogenesis, the de novo formation of blood vessels from precursor cells is critical for a developing embryo. However, the signals and events that dictate the formation of primary axial vessels remain poorly understood. Methodology/Principal Findings In this study, we use ets-related protein-1 (etsrp), which is essential for vascular development, to analyze the early stages of vasculogenesis in zebrafish. We found etsrp + cells of the head, trunk and tail follow distinct developmental sequences. Using a combination of genetic, molecular and chemical approaches, we demonstrate that fli + etsrp + hemato-vascular progenitors (FEVPs) are proliferating at the lateral plate mesoderm (LPM). The Shh-VEGF-Notch-Hey2 signaling pathway controls the proliferation process, and experimental modulation of single components of this pathway alters etsrp + cell numbers at the LPM. Conclusions/Significance This study for the first time defines factors controlling proliferation, and cell numbers of pre-migratory FEVPs in zebrafish.